Due to their highly stable nature in blood and relatively few side effects, antibodies have been receiving much attention as pharmaceuticals. Of particular note are bispecific antibodies that can simultaneously recognize two types of antigens. MDX-210, which is currently under clinical trial investigation, is an IgG-type bispecific antibody that retargets FcγRI-expressing monocytes and such to HER-2/neu-expressing cancer cells (see Non-Patent Document 1). In general, antibodies are produced using genetic recombination techniques. One specific technique involves the cloning of a DNA encoding an antibody protein from antibody-producing cells, such as hybridomas or sensitized lymphocytes that produce antibodies or a phage library presenting antibody genes, and the insertion of such into a suitable vector, which is then transfected into host cells for antibody production. Production of IgG type bispecific antibodies using genetic recombination techniques involves the introduction of a total of four types of genes into cells, in which these genes of H chains and L chains constitute two types of IgGs of interest, and the secretion of the antibodies by coexpression. In this type of system, expression of the wild type H chains and L chains constituting genes leads to random covalent bonding between two types of H chains and non-covalent bonding between H and L chains, and thus, the proportion of the bispecific antibody of interest becomes very small. More particularly, only one out of ten types produced is the bispecific antibody of interest, rendering the production efficiency quite low. Decreased efficiency in the production of the antibody of interest is not only an obstacle for purifying the antibody of interest, but also increases the nonuniformity, such as the lot-to-lot differences, which, in turn, leads to swelling production costs.
Preferential secretion of IgGs with a heterologous combination of H chains by introducing amino acid substitutions into the IgG H chain CH3 region has been reported as a means to improve the efficiency of bispecific antibody production (see Patent Document 1 and Non-Patent Documents 2 and 3). This method involves induction of promotion of heterologous H chain formation and inhibition of homogeneous H chain formation by substituting an amino acid side chain present in the CH3 region of one of the H chains to a larger side chain (knob), and substituting the amino acid side chain present in the CH3 region of the other H chain to a smaller side chain (hole), such that the knob is placed into the hole. A finding that uses a similar “knob” and “hole” at the interface where the H chain variable region (hereinafter referred to as VH) associates with the L chain variable region (hereinafter referred to as VL) has also been reported (see Non-Patent Document 4). According to the report by Zhe et al., substitution of two types of amino acids present at the VH-VL interface (four types for both chains) promotes the formation of the heterologous molecule 1.28 times more efficiently (wild type: 72%, and modified type: 92%). Meanwhile, substitution of one type of amino acid (two types for both chains) results in the same level of efficiency as the wild type. However, the method of setting a knob and a hole in VH and VL does not sufficiently promote the formation of heterologous molecules.    [Patent Document 1] International publication WO 96/27011    [Non-Patent Document 1] Segal D M et al., Current Opinion in immunology, 1999, Vol. 11, p. 558-562.    [Non-Patent Document 2] Ridgway J B et al., Protein Engineering, 1996, Vol. 9, p. 617-621.    [Non-Patent Document 3] Merchant A M et al., Nature Biotechnology, 1998, Vol. 16, p. 677-681.    [Non-Patent Document 4] Zhe Z et al., Protein Science, 1997, Vol. 6, p. 781-788.